Lens is a transparent tissue that lacks vasculature. Transmembrane water channels known as aquaporins (AQPs) play a significant role in maintaining transparency and homeostasis in the avascular lens. Mutations in AQP0 result in lens cataract in both human and mouse; knockout leads to cataract in mouse. Our long term goal is to contribute to the treatment and prevention of lens cataract. In this proposal, we are directing our focus on AQP0. At least two functions have been attributed for AQP0 viz., water permeability and cell-to-cell adhesion. Water permeability has been proven authentically through in vitro and in vivo studies while cell-to-cell adhesion function remains hypothetical; moreover, it is controversial whether intact as well as cleaved forms of AQP0 function both as a water pore and a cell-to-cell adhesion protein. The goals of this proposal is to critically experiment and clearly define whether AQP0 functions as a cell-to-cell adhesion protein, whether intact as well as the N- or and C- terminus cleaved forms function both as a water pore and a cell-to-cell adhesion protein (Aim 1), whether the extracellular loops play a critical role in cell-to-cell adhesion (Aim 2), whether the calmodulin-binding domain has a role with regard to adhesion function (Aim 3) and whether the function/s of AQP0 can be replaced by knocking in AQP4 which is proven to have both water permeability and cell-to-cell adhesion function (Aim 4). A novel method developed will be used to study the cell-to-cell adhesion function of the different forms of AQP0. Involvement of extracellular loop in cell-to-cell adhesion and the role of calmodulin binding domain for the functions of AQP0 will be studied using site-directed mutagenesis and PCR-based domain swapping. Computer based molecular simulation will be performed as necessary for mutagenesis studies. Knock-in animal models will be developed to test the in vivo outcome of the in vitro findings. The objectives will be pursued using structure-function approach and performing cytological, biochemical and molecular biological experiments as appropriate to verify the results. PUBLIC HEALTH RELEVANCE: Lens cataract is responsible for majority of the loss of sight in the United States and ranks #1 globally with over 20 million people around the world already blinded and an additional 1.25 million added to the total each year; age-related cataract by itself accounts for more than 48% of loss of vision scenarios. Lens cataract caused by AQP0 falls in the category of conformational diseases (proteopathies) as they are mainly caused by missense mutations or age related protein misfolding, aggregation and cytotoxicity and main goal of this project is to provide new insights into the structure-related functional roles of AQP0, the most abundant integral membrane protein in the lens, for maintaining transparency and homeostasis. This investigation will contribute to an improved understanding of normal lens development and aging processes, and help to design and develop therapeutic compounds/drugs for congenital and senile cataracts in humans.